Since 2003, as the Human Genome Project develops, the first generation sequencing (Sanger) and the second generation sequencing (NGS) techniques have been gradually occupying the molecular diagnostics market due to their sensitivity and accuracy. Genomics at the molecular level has also become a research hotspot in biological sciences for the 21st century. The emerging clinical application of gene sequencing technologies, the discovery of various cancer-related genes and the advancement of the Cancer Genome Project represent the dawn of early diagnosis, personalized therapy and prognosis of cancers. Currently, genomic DNA is primarily obtained from tissue cells by such processes as extraction following disruption of live tissue cells and in vitro PCR amplification from paraffin specimen of tissue cells. However, there still exists a technical barrier as to how to diagnose the onset of cancers and even prevent them at DNA level for early-stage patients.
The data obtained about the survival rate against malignancies provides important information that reflects tumor burden in a region and that can be used to evaluate the medical resources as well as the level of prevention and treatment in the region. An analysis of cancer survival rate during 2003 to 2005 conducted by National Center for Cancer Registries revealed that five-year relative survival rate was only 30.9% in our country. The survival rates for major cancers were 16.1% for lung cancer, 27.4% for stomach cancer, 10.1% for liver cancer, 20.9% for esophagus cancer, 47.2% for colorectal cancer, and 73% for breast cancer, respectively. The cancer survival rates differ significantly between urban and rural areas. While in developed countries, the five-year survival rates for colorectal cancer and breast cancer reached 60% and 85% respectively, and the rates for liver cancer and lung cancer with poor prognosis reached 15 to 20%, higher than in our country (data from Lancet). It is evident that “three earlies”, i.e., early discovery, early diagnosis and early treatment are required in order to raise the five-year survival rates of cancer patients in our country.
The means currently clinically used for diagnosing and evaluating cancers mainly involves a combination of localization by imaging and qualification by tumor markers. Imaging techniques include X ray (chest fluoroscopy, chest X-ray film, low dose CT), magnetic resonance imaging (MIR), radioactive substance (nuclide bone scanning), and PET-CX ray as a conventional means of screening is mainly used for determining the size and the position of a tumor. It needs to be used in combination with detection of tumor markers to achieve a definite diagnosis. MIR detects whether there exists tumor metastasis to the brain or the spinal cord, and nuclide bone scanning detects whether there exists tumor metastasis to bones. Both detection techniques are mainly used for diagnosing post-phase III tumors. PET (positron emission computed tomography), characterized by being non-traumatic, is at present the only technique for imaging function, metabolism and receptors in the manner of anatomical morphology. PET-CT, as a combination of PET and CT techniques for screening for tumor location and metastasis, is one of the best means clinically used for diagnosing tumors and guiding the treatment of same. However, such diagnosis methods are relatively costly and are not covered by medical insurances. Many patients have to abandon the use of these methods because they are unable to afford the expensive diagnostic fee. Imaging detection is mainly used for localizing malignancies. In order to define the type and the phase of a cancer, it is necessary to further evaluate tumor markers, whereby the cancer can be identified or diagnosed according to the biochemical or immunological characteristics of the tumor markers. A tumor marker is a substance produced and released by a tumor cell, and is generally present in the tumor cell or in the body fluid of the host in the form of a metabolic product such as antigen, enzyme, hormone etc. Tumor markers useful in clinical detection are alpha-fetal protein (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen family (CA125, CA15-3 etc.), among others.
However, at stages when cancers are definitely diagnosed, the malignancies detectable by the clinically used conventional imaging techniques described above have a diameter of 1 cm or more, a tumor cell number on the order of 109, and a weight of more than 1 g. Despite its high sensitivity, PET-CT technique can only detect tumors larger than 0.5 cm or more (H. Li et al. 2013; Bu Zhaode, Xue Zhongqi et al., 2000). That is, tumor patients diagnosed by imaging techniques are mostly at the middle or late stage, and have already missed the optimal period of cancer treatment. At the early stage of a cancer, even if tumor markers are detected, the particular position of the tumor tissue cannot be identified by imaging detection and the tumor cannot be ultimately definitely diagnosed. Therefore the cancer cannot be treated, and can only be left to develop and worsen.
Drug resistance of tumors and recurrence after cure are among the most important reasons responsible for the death of tumor patients. Addressing these two problems has become a research hotspot in tumor diseases now in the world. Presently, there lacks an effective means for the evaluation of drug administration during clinical cancer treatment. Taking lung cancer as an example, gefitinib (Iressa) and Erlotinib (Tarceva) are drugs for treating non-small cell lung cancer (NSCLC), but clinical data suggested that such drugs are not suitable for use with all patients of non-small cell lung cancer. Further investigations found that patients with EGFR (epidermal growth factor receptor) mutation were more responsive to treatment with Iressa by 40% or higher than non-selective patients (Tony S. Mok, M. D. et al., 2009; Hida T, Okamoto I, Kashii T, et al., 2008; Kimura H, Kasahara K, Kawaishi M, et al., 2006). Gemcitabine is a difluoronucleoside antimetabolite anti-cancer drug that disrupts cell replication. It is suitable for treating non-small cell lung cancer at the middle and late stages. Researches indicated that patients with a low expression of RRM1 (RRM1 gene is mapped on the short arm of chromosome 1 and encodes ribonucleotide reductase M1 subunit) exhibited a control rate of the disease of 30% or higher when treated with gemcitabine (Lee et al., 2010). European Medicines Agency definitely specifies that patients with non-small cell lung cancer must be detected for EGFR gene before using Iressa, and patients with metastatic large intestine cancer must be detected for KRAS gene before using the targeting drugs Erbitux and Vectibix (see the official website of European Medicines Agency http://www.ema.europa.eu/ema/). It is evident that for the treatment of malignancies, guidance and evaluation of drug administration is an important precondition and basis for optimizing therapeutic regimens and achieving effective treatment. Studies on tumor onset and development by systems biology concluded that DNA mutation or genetically-acquired defective genes are key to the development of drug resistance. Therefore it is a general trend to provide, at gene level, personalized guidance and evaluation of drug administration.
In addition to the above, there exists a blind period of detection in the prognosis of clinical treatment of cancer. For example, many cancer patients have their tumor tissues removed by surgery to prevent the tissues from metastasizing and endangering their lives. After the surgery, they need to receive regular biopsy reexamination for prognostic evaluation. Due to the considerable radiation in imaging detection that does harm to human body, the cancer patients mostly receive reexamination post surgery on a yearly basis, which greatly affects the timeliness of prognostic evaluation. This suggests that there is an urgent need in the cancer diagnosis field for a more convenient and non-traumatic detection technique. In a word, the screening, diagnosis, treatment and prognosis of early-stage cancers in current clinical medicine would be a major breakthrough in cancer treatment, and would be key to increasing the five-year survival rates against the cancers. There are substantial technical problems in this field that confronts both medical research and clinical application activities.
In 1947, Mandel and Metais discovered an extracellular DNA in body fluid such as blood, synovial fluid and cerebrospinal fluid, which mainly exists in the form of a DNA-protein complex or a free DNA (Mandel and Metais, 1947). In the 1980's, Leon et al. found that the DNA level in the peripheral serum of tumor pateints was considerably higher than that in normal people (Leon et al., 1977). Moreover, researches revealed that cancer gene mutations consistent with primary tumors were detected in the plasma and serum of patients with the tumors. This suggests that circulating DNA as a novel tumor maker would play an important role in the diagnosis, treatment and prognosis of tumors. Circulating tumor DNA (ctDNA) is a DNA that is released into the circulation system after it comes off of a tumor cell or after apoptosis of the cell, and, as such, can be qualified, quantitated and traced. Successful capture of ctDNA and accurate interpretation of the information contained therein would provide an exact means for acquisition of gene information of early-stage cancers, early diagnosis and prognostic detection of the cancers, and evaluation of drug resistance.
However, such techniques are not effectively developed and widely used for the time being. One of the reasons is that ctDNA is present in the peripheral blood in a very low amount, and in particular, its relative content is extremely low in comparison to normal DNA (nDNA). It is still difficult for current detection technologies to directly detect the ctDNA level in peripheral blood. Therefore, a need exists to enrich target ctDNA by using a DNA enriching process prior to sequencing the enriched ctDNA to obtain the information contained therein and ascertain the diseased state of a subject according to the information obtained from sequencing.